Site-directed spin-labeling reveals the orientation of the amino acid side-chains in the E-F loop of bacteriorhodopsin

Due to high temperature factors and the lack of considerable electron densi ty, electron microscopy and X-ray experiments on the cytoplasmic E-F loop o f bacteriorhodopsin result in a variety of structural models. As the experi mental conditions regarding ionic strength, temperature and the presence of detergents may affect the structure of the E-F loop, we employ electron pa ramagnetic resonance and site-directed spin-labeling to study the structure of this loop under physiological conditions. The amino acid residues at po sitions 154 to 171 were replaced by cysteine residues and derivatized with a sulfhydryl-specific nitroxide spin label one by one. The conventional and power saturation electron paramagnetic spectroscopy provide the mobility o f the nitroxide and its accessibility to dissolved molecular oxygen and mem brane-impermeable chromium oxalate in the respective site. The results show that K159 and A168 are located at the water-lipid interface of helices E a nd F, respectively. The orientation of the amino acid side-chains in the he lical regions from positions 154 to 159 and 166 to 171 were found to agree with published structural data for bacteriorhodopsin. In the residue sequen ce from positions 160 to 165 the EPR data yield evidence for a turned loop structure with the side-chains of M163 and S162 oriented towards the proton channel and the water phase, respectively. (C) 1999 Academic Press.